Method of identifying bitter compounds by employing TRP8, a transient receptor potential channel expressed in taste receptor cells

ABSTRACT

The present invention relates to the discovery, identification and characterization of a transient receptor potential channel, referred to herein as TRP8, which is expressed in taste receptor cells and associated with the perception of bitter and sweet taste. The invention encompasses TRP8 nucleotides, host cell expression systems, TRP8 proteins, fusion proteins, polypeptides and peptides, antibodies to the TRP8 protein, transgenic animals that express a TRP8 transgene, and recombinant “knock-out” animals that do not express TRP8. The invention further relates to methods for identifying modulators of the TRP8-mediated taste response and the use of such modulators to either inhibit or promote the perception of bitterness or sweetness. The modulators of TRP8 activity may be used as flavor enhancers in foods, beverages and pharmaceuticals.

This Application claims priority under 35 U.S.C 119(e) to U.S.application No. 60/197,491 filed Apr. 17, 2000.

1. INTRODUCTION

The present invention relates to the discovery, identification andcharacterization of a transient receptor potential channel, referred toherein as TRP8, which is expressed in taste receptor cells andassociated with the perception of bitter and sweet taste. The inventionencompasses TRP8 nucleotides, host cell expression systems, TRP8proteins, fusion proteins, polypeptides and peptides, antibodies to theTRP8 protein, transgenic animals that express a TRP8 transgene, andrecombinant “knock-out” animals that do not express TRP8. The inventionfurther relates to methods for identifying modulators of theTRP8-mediated taste response and the use of such modulators to eitherinhibit or promote the perception of bitterness or sweetness. Themodulators of TRP8 activity may be used as flavor enhancers in foods,beverages and pharmaceuticals.

2. BACKGROUND OF THE INVENTION

Mammals are generally thought to have five basic categories of tasteperception: salt, sour, sweet, bitter and umami (monosodium glutamate)(for review, see Lindemann, 1996, Physiological Reviews, 76:719-766;Herness and Gilbertson, 1999, Annu Rev. Physiol., 61:873:900). The tastesignals are sensed by specialized taste receptor cells (TRCs), which areorganized into taste buds. Each taste bud comprises between about 50 and100 individual cells grouped into a cluster that is between 20 and 40microns in diameter. Nerve fibers enter from the base of the taste budand synapse onto some of the taste receptor cells. Typically, a singleTRC contacts several sensory nerve fibers, and each sensory fiberinnervates several TRCs in the same taste bud (Lindemann, supra).

TRCs of most, if not all, vertebrate species possess voltage-gatedsodium, potassium, and calcium ion channels with properties similar tothose of neurons (Kinnamon, S. C. & Margolskee, R. F., 1996, Curr. Opin.Neurobiol. 6:506-513). Different types of primary tastes appear toutilize different types of transduction mechanisms, and certain types oftastes may employ multiple mechanisms which may reflect varyingnutritional requirements amongst species (Kinnamon & Margolskee, supra).

Bitter and sweet taste transduction are thought to involve cAMP and IP₃(Kinnamon & Margolskee, supra). The bitter compound denatonium causescalcium ion release from rat TRCs and the rapid elevation of IP₃ levelsin rodent taste tissue (Id., citing Bernhardt, S J. et al., 1996, J.Physiol. (London) 490:325-336 and Akabas, M. H., et al., 1988, Science242:1047-1050). Since denatonium cannot pass the cell membrane, it hasbeen suggested that it may activate G-protein-coupled receptors, wherebythe α and/or βγ G protein subunits would activate phospholipase C,leading to IP₃ generation and the release of calcium ions (Kinnamon &Margolskee, supra).

In recent years, a taste-specific G protein termed “gustducin”, which ishomologous to the retinal G protein, transducin, has been cloned andcharacterized (Id., citing McLaughlin, S. et al., 1992, Nature (London)357:563-569). It is believed that gustducin plays a direct role in bothbitter and sweet transduction. For example, gustducin and subunit(∝-gustducin) null (knockout) mice had a reduced aversion to bittercompounds. Unexpectedly, the mice also exhibited a preference for sweetcompounds suggesting involvement of gustducin in sweet transduction.

Recent biochemical experiments have demonstrated that taste receptorpreparations activate transducin and gustducin in response to denatoniumand other bitter compounds (Ming et al., 1998, Proc. Natl. Acad. Sci.USA 95:8933-8).

To thoroughly understand the molecular mechanisms underlying tastesensation, it is important to identify each molecular component in thetaste signal transduction pathways. The present invention relates to thecloning of an ion channel, TRP8 (transient receptor potential channel8), that is believed to be involved in taste transduction and may beinvolved in the changes in intra-cellular calcium ions associated withbitter taste perception.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification andcharacterization of a transient receptor potential (TRP) channel,referred to hereafter as TRP8, that participates in the taste signaltransduction pathway. TRP8 is a channel protein with a high degree ofstructural similarity to the family of calcium channel proteins known astransient receptor potential channels. As demonstrated by Northern Blotanalysis, expression of the TRP8 transcript is tightly regulated, withthe highest level of gene expression found in taste tissue, moderateexpression in stomach and small intestine, and very low level expressionin uterus and testis. In situ hybridization indicated expression of TRP8in circumvallate and foliate papillae, but not in the surroundingnon-gustatory epithelia. Additionally, the general pattern of TRP8expression was comparable to that of α-gustducin, although theα-gustducin signal was somewhat more intense.

The present invention encompasses TRP8 nucleotides, host cellsexpressing such nucleotides and the expression products of suchnucleotides. The invention encompasses TRP8 protein, TRP8 fusionproteins, antibodies to the TRP8 channel protein and transgenic animalsthat express a TRP8 transgene or recombinant knock-out animals that donot express the TRP8 protein.

Further, the present invention also relates to screening methods thatutilize the TRP8 gene and/or TRP8 gene products as targets for theidentification of compounds which modulate, i.e., act as agonists orantagonists, of TRP8 activity and/or expression. Compounds whichstimulate taste responses similar to those of bitter tastants can beused as additives to provoke a desired aversive response—for example todiscourage ingestion of compositions containing these compounds bychildren or animals. Compounds which inhibit the activity of the TRP8channel may be used to block the perception of bitterness. Theinhibitors of TRP8 may be used as flavor enhancers in foods, beveragesor pharmaceuticals by decreasing or eliminating the perception of bittertaste.

The invention is based, in part, on the discovery of a channel proteinexpressed at high levels in taste receptor cells. In taste transduction,bitter compounds are thought to act via the G-proteins, such asgustducin, which in turn regulate second messenger systems.Co-localization of α-gustducin, γ-gustducin, phospholipase Cβ₂ (PLCβ₂)and TRP8 to one subset of taste receptor cells indicates that they mayfunction in the same transduction pathway. It is believed that TRP8responds to tastant induced inositol triphosphate (IP₃)/diacylglycerol(DAG) generation by flooding the taste cell with extracellular calciumand activating calcium dependent down stream messengers leading totransmitter release into the synapse and activation of afferentgustatory nerves.

3.1. DEFINITIONS

As used herein, italicizing the name of TRP8 shall indicate the TRP8gene, in contrast to its encoded protein product which is indicated bythe name of TRP8 in the absence of italicizing. For example, “TRP8”shall mean the TRP8 gene, whereas “TRP8” shall indicate the proteinproduct of the TRP8 gene.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Nucleotide sequence of the murine TRP8 cDNA encoding murineTRP8, SEQ ID NO: 1.

FIG. 2. Deduced amino acid sequence of the murine TRP8 transientreceptor potential channel, SEQ ID NO: 2.

FIGS. 3A-B. Nucleotide sequence of the human TRP8 cDNA encoding humanTRP8, SEQ ID NO: 3.

FIG. 4. Deduced amino acid sequence of the human TRP8 protein transientreceptor potential channel, SEQ ID NO: 4.

FIG. 5. Amino acid sequence of the murine TRP8 (upper lines);

-   -   versus human TRP8 (lower lines), as represented in part by SEQ        ID NO: 2 and SEQ ID NO: 4, respectively, and displayed in SEQ ID        NO: 6. Each pair of lines corresponds to a predicted mouse/human        exon.

FIGS. 6A-C. Predicted topography of the TRP8 protein transient receptorpotential channel in the membrane.

FIG. 7. Distribution of TRP8 mRNA and protein in mouse tissues. (a)Autoradiogram of a northern blot hybridized with a TRP8 cDNA probe. Eachlane contained 25 μg total RNA isolated from the following mousetissues: circumvallate and foliate papillae-enriched taste tissue (Tastetissue), lingual tissue devoid of taste buds (Non-taste), brain, retina,olfactory epithelium (Olf. Epi.), stomach, small intestine (Small Int.),thymus, heart, lung, spleen, skeletal muscle (Skele. Mus.), liver,kidney, uterus and testis. A 4.5 kb transcript was detected in tastetissue, stomach and small intestine, and to a much lesser extent, inuterus and testis. To control for MRNA quantity the same blot wasstripped and reprobed with a β-actin cDNA probe (lower panel). The sizein kilobases (kb) of RNA markers is indicated at the right-hand side.(b) Autoradiogram of a western blot probed with an anti-TRP8 antibody.Protein extracts (50 μg) prepared from the murine tissues indicated wereelectrophoresed, transferred to a nitrocellulose membrane, then the blotincubated with an antibody against the carboxyl-terminal of TRP8.Immunoreactive protein of ˜130 kD, the predicted molecular weight ofTRP8, was detected in stomach and small intestine; a higher molecularweight species was identified in liver and kidney. Molecular sizemarkers are given in kilodaltons.

FIG. 8. TRP8 mRNA is expressed in taste receptor cells. Sections ofmurine lingual epithelia containing circumvallate and foliate papillaewere hybridized with ³³P-labeled antisense RNA probes for TRP8 (a,c) andα-gustducin (d), and subjected to autoradiography. Photomicrographs ofcircumvallate (a) and foliate (b) papillae hybridized to the antisenseTRP8 probe demonstrates expression of TRP8 in a subset of TRCs. (d)Shows hybridization of an α-gustducin antisense probe to foliatepapillae. Hybridization controls with sense probes showed the absence ofnon-specific binding of the TRP8 probe (b) or the α-Gustducin probe (e).

FIG. 9. Co-localization in TRCs of TRP8 and other signal transductionelements. Immunofluorescence of Gγ13 (a) and TRP8 (b) in the samelongitudinal section of mouse taste papillae section: (c) is the overlayof a and b. Immunofluorescence of TRP8 (d) and α-gustducin (e) in thesame section: (f) is the overlay of d and e. Immunofluorescence of PLCβ2(g) and TRP8 (h) in the same section: (i) is the overlay of g and h.

FIG. 10. Profiling the pattern of expression of TRP8, a-gustducin, Gβ1,Gβ3, Gγ13 and PLCβ2 in taste tissue and taste cells. Left panel:Southern hybridization to RT-PCR products from murine taste tissue (T)and control non-taste lingual tissue (N). 3′-region probes from TRP8,α-gustducin (Gust), Gβ1, Gβ3, Gγ13, PLCβ2 and glyceraldehyde 3-phosphatedehydrogenase (G3PDH) were used to probe the blots. Note that TRP8,α-gustducin, Gβ1, Gβ3, Gγ13 and PLCβ2 were all expressed in tastetissue, but not in non-taste tissue. Right panel: Southern hybridizationto RT-PCR products from 24 individually amplified taste receptor cellsfrom a transgenic mouse expressing green fluorescent protein (GFP) fromthe gustducin promoter. 19 cells were GFP-positive (+), 5 cells wereGFP-negative (−). Expression of TRP8, α-gustducin, Gβ3, Gγ13and PLCβ2was fully coincident. 15 of 19 TRP8-positive cells were also positivefor Gβ1. G3PDH served as a positive control to demonstrate successfulamplification of products.

FIG. 11. TRP8, but not mTrp 1-7, is detected by PCR in taste tissue. PCRamplifications of TRP8 and mTrp 1-7 were performed using non-degenerateprimers specific for each Trp family member. Taste cDNA (upper panels)and brain cDNA (lower panels) provided templates for amplification.Amplified material was resolved in a 1.2% agarose gel. Bands of theexpected molecular weight were sequenced to verify the identity of theTrp channel amplified. Positive (G3PDH primers) and negative (noprimers) controls are shown (right panels).

FIG. 12. Heterologous expression of TRP8. Xenopus oocytes were injectedwith 50 ng of TRP8 cRNA (a) or 50 nl of water (b); two days afterinjection, oocytes were treated with thapsigargin (2 μM), followed bythe addition of Ca++(10 mM) or EGTA as indicated (arrows). The tracesrepresent currents induced at negative membrane potentials (commandvoltage −80 mV). (c) I-V curve for oocytes injected with TRP8 cRNA orwater demonstrates a reversal potential, consistent with Ca++ activationof the endogenous calcium-activated chloride conductance (ICl_(Ca)). (d)The maximal inward current elicited with external Ca++ present in thebathing media for oocytes injected with TRP8 cRNA or water (control).

FIG. 13. TRP8 functions as a Ca++ channel. Xenopus oocytes were injectedwith 50 ng of TRP8 cRNA (right panels) or 50 nl of water (left panels);two days after injection, oocytes were treated with thapsigargin (2 μM),followed by the addition of Ca++ (10 mM).

FIG. 14. Potential signal transduction pathways in TRCs utilizing TRP8.Responses to bitter compounds such as denatonium are initiated bybinding to one or more gustducin-coupled receptors of the T2R/TBRfamily. Activation of the gustducin heterotrimer releases its βγmoiety(e.g. Gβ3/Gγ13) which stimulates PLCβ2, resulting in production of IP₃and DAG. IP₃ binds to its receptors e.g. IP₃R3 and causes the release ofCa⁺⁺ from intracellular stores, triggering activation of TRP8 channels,which ultimately leads to the influx of Ca⁺⁺ through TRP8 channels. DAGmay act directly on TRP8 to lead to Ca⁺⁺ influx. Artificial sweetenersmay depend on a similar transduction pathway, but with sweet-responsivereceptors, e.g., T1R3 coupled to gustducin or other G proteinsinitiating the signal that leads to the production of IP₃ and DAG andstimulation of TRP8.

5. DETAILED DESCRIPTION OF THE INVENTION

TRP8 is a channel protein that participates in receptor-mediated tastesignal transduction and belongs to the family of calcium channelproteins known as transient receptor potential channels (Montell C.,1997, Mol. Pharmacol. 52:755-763) The present invention encompasses TRP8nucleotides, TRP8 proteins and peptides, as well as antibodies to theTRP8 protein. The invention also relates to host cells and animalsgenetically engineered to express the TRP8 channel or to inhibit or“knock-out” expression of the animal's endogenous TRP8.

The invention further provides screening assays designed for theidentification of modulators, such as agonists and antagonists, of TRP8activity. The use of host cells that naturally express TRP8 orgenetically engineered host cells and/or animals offers an advantage inthat such systems allow the identification of compounds that affect thesignal transduced by the TRP8 protein.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1. THE TRP8 GENE

The cDNA sequence and deduced amino acid sequence of murine TRP8 areshown in FIGS. 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively. ThecDNA and deduced ammo acid sequence of human TRP8 are shown in FIGS. 3(SEQ ID NO: 3) and 4 (SEQ ID NO: 4), respectively.

The TRP8 nucleotide sequences of the invention include: (a) the DNAsequences shown in FIG. 1 (SEQ ID NO: 1) or 3 (SEQ ID NO: 3) orcontained in the cDNA clone pMR24 within E. coli strain XL10 Gold asdeposited with the American Type Culture Collection (ATCC Accession No.); (b) nucleotide sequences t,hat encode the amino acid sequence shownin FIG. 2 (SEQ ID NO: 2) or 4 (SEQ ID NO: 4) or the TRP8 amino acidsequence encoded by the cDNA clone pMR24 as deposited with the ATCC; (c)any nucleotide sequence that (i) hybridizes to the nucleotide sequenceset forth in (a) or (b) under stringent conditions, e.g., hybridizationto filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1mM EDTA at 65EC, and washing in 0.1×SSC/0.1% SDS at 68EC (Ausubel F.M.et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,Green Publishing Associates, Inc., and John Wiley & sons, Inc., NewYork, at p. 2.10.3) and (ii) encodes a functionally equivalent geneproduct; and (d) any nucleotide sequence that hybridizes to a DNAsequence that encodes the amino acid sequence shown in FIG. 1 (SEQ IDNO: 1) or 3 (SEQ ID NO: 3), or that is contained in cDNA clone pMR24 asdeposited with the ATCC, under less stringent conditions, such asmoderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at42EC (Ausubel et al., 1989 supra), yet which still encodes afunctionally equivalent TRP8 gene product. Functional equivalents of theTRP8 protein include naturally occurring TRP8 present in species otherthan mice and humans. The invention also includes degenerate variants ofsequences (a) through (d). The invention also includes nucleic acidmolecules, that may encode or act as TRP8 antisense molecules, useful,for example, in TRP8 gene regulation (for and/or as antisense primers inamplification reactions of TRP8 gene nucleic acid sequences).

In addition to the TRP8 nucleotide sequences described above, homologsof the TRP8 gene present in other species can be identified and readilyisolated, without undue experimentation, by molecular biologicaltechniques well known in the art. For example, cDNA libraries, orgenomic DNA libraries derived from the organism of interest can bescreened by hybridization using the nucleotides described herein ashybridization or amplification probes.

The invention also encompasses nucleotide sequences that encode mutantTRP8s, peptide fragments of the TRP8, truncated TRP8, and TRP8 fusionproteins. These include, but are not limited to nucleotide sequencesencoding polypeptides or peptides corresponding to the TM(transmembrane) and/or CD (cytoplasmic) domains of TRP8 or portions ofthese domains; truncated TRP8s in which one or two of the domains isdeleted, e.g., a functional TRP8 lacking all or a portion of the CDregion. Certain of these truncated or mutant TRP8 proteins may act asdominant-negative inhibitors of the native TRP8 protein. Nucleotidesencoding fusion proteins may include but are not limited to full lengthTRP8, truncated TRP8 or peptide fragments of TRP8 fused to an unrelatedprotein or peptide such as an enzyme, fluorescent protein, luminescentprotein, ect., which can be used as a marker.

TRP8 nucleotide sequences may be isolated using a variety of differentmethods known to those skilled in the art. For example, a cDNA libraryconstructed using RNA from a tissue known to express TRP8 can bescreened using a labeled TRP8 probe. Alternatively, a genomic librarymay be screened to derive nucleic acid molecules encoding the TRP8channel protein. Further, TRP8 nucleic acid sequences may be derived byperforming PCR using two oligonucleotide primers designed on the basisof the TRP8 nucleotide sequences disclosed herein. The template for thereaction may be cDNA obtained by reverse transcription of mRNA preparedfrom cell lines or tissue known to express TRP8.

The invention also encompasses (a) DNA vectors that contain any of theforegoing TRP8 sequences and/or their complements (i.e., antisense); (b)DNA expression vectors that contain any of the foregoing TRP8 sequencesoperatively associated with a regulatory element that directs theexpression of the TRP8 coding sequences; (c) genetically engineered hostcells that contain any of the foregoing TRP8 sequences operativelyassociated with a regulatory element that directs the expression of theTRP8 coding sequences in the host cell; and (d) transgenic mice or otherorganisms that contain any of the foregoing TRP8 sequences. As usedherein, regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

5.2. TRP8 PROTEINS AND POLYPEPTIDES

TRP8 protein, polypeptides and peptide fragments, mutated, truncated ordeleted forms of the TRP8 and/or TRP8 fusion proteins can be preparedfor a variety of uses, including but not limited to the generation ofantibodies, the identification of other cellular gene products involvedin the regulation of TRP8 mediated taste perception, and the screeningfor compounds that can be used to modulate taste perception such asbitter blocking agents and taste modifiers.

FIGS. 2 (SEQ ID NO: 2) and 4 (SEQ ID NO: 4) show the deduced amino acidsequence of the murine and human TRP8 protein, respectively. The TRP8amino acid sequences of the invention include the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2) or FIG. 4 (SEQ ID NO: 4), or the aminoacid sequence encoded by cDNA clone pMR24 as deposited with the ATCC.Further, TRP8s of other species are encompassed by the invention. Infact, any TRP8 protein encoded by the TRP8 nucleotide sequencesdescribed in Section 5.1, above, is within the scope of the invention.

The invention also encompasses proteins that are functionally equivalentto the TRP8 encoded by the nucleotide sequences described in Section5.1, as judged by any of a number of criteria, including but not limitedto the ability of a bitter tastant to trigger the influx of calcium fromextracellular calcium stores into a taste receptor cell expressing saidprotein, leading to transmitter release from the taste receptor cellinto the synapse and activation of an afferent nerve. Such functionallyequivalent TRP8 proteins include but are not limited to proteins havingadditions or substitutions of amino acid residues within the amino acidsequence encoded by the TRP8 nucleotide sequences described, above, inSection 5.1, but which result in a silent change, thus producing afunctionally equivalent gene product.

Peptides corresponding to one or more domains of TRP8 (e.g.,transmembrane (TM) or cellular domain (CD)), truncated or deleted TRP8s(e.g., TRP8 in which the TM and/or CD is deleted) as well as fusionproteins in which the full length TRP8, a TRP8 peptide or a truncatedTRP8 is fused to an unrelated protein are also within the scope of theinvention and can be designed on the basis of the TRP8 nucleotide andTRP8 amino acid sequences disclosed herein. Such fusion proteins includefusions to an enzyme, fluorescent protein, or luminescent protein whichprovide a marker function.

While the TRP8 polypeptides and peptides can be chemically synthesized(e.g, see Creighton, 1983, Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co., N.Y.), large polypeptides derived fromTRP8 and the full length TRP8 itself may be advantageously produced byrecombinant DNA technology using techniques well known in the art forexpressing a nucleic acid containing TRP8 gene sequences and/or codingsequences. Such methods can be used to construct expression vectorscontaining the TRP8 nucleotide sequences described in Section 5.1 andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. (See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra).

A variety of host-expression vector systems may be utilized to expressthe TRP8 nucleotide sequences of the invention. Where the TRP8 peptideor polypeptide is expressed as a soluble derivative (e.g., peptidescorresponding to TM and/or CD) and is not secreted, the peptide orpolypeptide can be recovered from the host cell. Alternatively, wherethe TRP8 peptide or polypeptide is secreted the peptide or polypeptidesmay be recovered from the culture media. However, the expression systemsalso include engineered host cells that express TRP8 or functionalequivalents, anchored in the cell membrane. Purification or enrichmentof the TRP8 from such expression systems can be accomplished usingappropriate detergents and lipid micelles and methods well known tothose skilled in the art. Such engineered host cells themselves may beused in situations where it is important not only to retain thestructural and functional characteristics of the TRP8, but to assessbiological activity, i.e., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors containing TRP8 nucleotide sequences; yeasttransformed with recombinant yeast expression vectors containing TRP8nucleotide sequences or mammalian cell systems harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells or from mammalian viruses.

Appropriate expression systems can be chosen to ensure that the correctmodification, processing and sub-cellular localization of the TRP8channel protein occurs. To this end, eukaryotic host cells which possessthe ability to properly modify and process the TRP8 channel protein arepreferred. For long-term, high yield production of recombinant TRP8channel protein, such as that desired for development of cell lines forscreening purposes, stable expression is preferred. Rather than usingexpression vectors which contain origins of replication, host cells canbe transformed with DNA controlled by appropriate expression controlelements and a selectable marker gene, i.e., tk, hgprt, dhfr, neo, andhygro gene, to name a few. Following the introduction of the foreignDNA, engineered cells may be allowed to grow for 1-2 days in enrichedmedia, and then switched to a selective media. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that modulate the endogenous activity of the TRP8 geneproduct.

5.3. TRANSGENIC ANIMALS

The TRP8 gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, monkeys, and chimpanzees may be used to generate TRP8transgenic animals.

Any technique known in the art may be used to introduce the TRP8transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell,56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry theTRP8 transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal., (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236).The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art. When it is desired thatthe TRP8 transgene be integrated into the chromosomal site of theendogenous TRP8 gene, gene targeting is preferred. Briefly, when such atechnique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous TRP8 gene are designed for thepurpose of integrating, via homologous recombination with chromosomalsequences, into and disrupting the function of the nucleotide sequenceof the endogenous TRP8 gene.

Once transgenic animals have been generated, the expression of therecombinant TRP8 gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of TRP8 gene-expressing tissue may also beevaluated immunocytochemically using antibodies specific for the TRP8transgene product.

5.4. ANTIBODIES TO TRP8 PROTEINS

Antibodies that specifically recognize one or more epitopes of TRP8, orepitopes of conserved variants of TRP8, or peptide fragments of TRP8 arealso encompassed by the invention. Such antibodies include but are notlimited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention may be used, for example, in conjunctionwith compound screening schemes, as described, below, in Section 5.5,for the evaluation of the effect of test compounds on expression and/oractivity of the TRP8 gene product.

For production of antibodies, various host animals may be immunized byinjection with a TRP8 protein, or TRP8 peptide. Such host animals mayinclude but are not limited to rabbits, mice, and rats, to name but afew. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such asBCG-(Bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies comprising heterogeneous populations of antibodymolecules, may be derived from the sera of the immunized animals.Monoclonal antibodies may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-9).Such antibodies may be of any immunoglobulin class including IgG, IgM,IgE, IgA, IgD and any subclasses thereof. The hybridoma producing themAb of this invention may be cultivated in vitro or in vivo. Productionof high titres of Mabs in vivo makes this the presently preferred methodof production.

In addition, techniques developed for the production of “chimericantibodies” by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used(Morrison et al., 1984, Proc. Nat'l. Acad. Sci., 81:6851-6855; Neubergeret al., 1984, Nature, 312: 604-608; Takeda et al. 1985, Nature 314:452-454). Alternatively, techniques developed for the production ofhumanized antibodies (U.S. Pat. No. 5,585,089) or single chainantibodies (U.S. Pat. No. 4,946,778 Bird, 1988, Science 242: 423-426;Huston et al., 1988, Proc. Nat'l. Acad. Sci USA, 85: 5879-5883; and Wardet al., 1989, Nature 334: 544-546) may be used to produce antibodiesthat specifically recognize one or more epitopes of TRP8.

5.5. SCREENING ASSAYS FOR DRUGS AND OTHER CHEMICAL COMPOUNDS USEFUL INREGULATION OF TASTE PERCEPTION

The present invention relates to screening assay systems designed toidentify compounds or compositions that modulate TRP8 activity or TRP8gene expression, and thus, may be useful for modulation of bitter tasteperception.

In accordance with the invention, a cell-based assay system can be usedto screen for compounds that modulate the activity of the TRP8 andthereby, modulate the perception of bitterness. To this end, cells thatendogenously express TRP8 can be used to screen for compounds.Alternatively, cell lines, such as 293 cells, COS cells, CHO cells,fibroblasts, and the like, genetically engineered to express TRP8 can beused for screening purposes. Preferably, host cells geneticallyengineered to express a functional TRP8 are those that respond toactivation by bitter tastants, such as taste receptor cells. Further,ooyctes or liposomes engineered to express the TRP8 channel protein maybe used in assays developed to identify modulators of TRP8 activity.

The present invention provides for methods for identifying a compoundthat induces the perception of a bitter taste (a “bitterness activator”)comprising (i) contacting a cell expressing the TRP8 channel proteinwith a test compound and measuring the level of TRP8 activation; (ii) ina separate experiment, contacting a cell expressing the TRP8 channelprotein with a vehicle control and measuring the level of TRP8activation where the conditions are essentially the same as in part (i),and then (iii) comparing the level of activation of TRP8 measured inpart (i) with the level of activation of TRP8 in part (ii), wherein anincreased level of activated TRP8 in the presence of the test compoundindicates that the test compound is a TRP8 activator.

The present invention also provides for methods for identifying acompound that inhibits the perception of a bitter taste (a “bitternessinhibitor”) comprising (i) contacting a cell expressing the TRP8 channelprotein with a test compound in the presence of a bitter tastant andmeasuring the level of TRP8 activation; (ii) in a separate experiment,contacting a cell expressing the TRP8 channel protein with a bittertastant and measuring the level of TRP8 activation, where the conditionsare essentially the same as in part (i) and then (iii) comparing thelevel of activation of TRP8 measured in part (i) with the level ofactivation of TRP8 in part (ii), wherein a decrease level of activationof TRP8 in the presence of the test compound indicates that the testcompound is a TRP8 inhibitor.

A “bitter tastant”, as defined herein, is a compound or molecularcomplex that induces, in a subject, the perception of a bitter taste. Inparticular, a bitter tastant is one which results in the activation ofthe TRP8 channel protein resulting in an influx of Ca⁺² into the cell.Examples of bitter tastants include but are not limited to denatoniumbenzoate (“denatonium”; also “DEN”), quinine hydrochloride (“quinine”;also “QUI”), strychnine hydrochloride (“strychnine”; also “STR”),nicotine hemisulfate (“nicotine”; also “NIC”), atropine hydrochloride(“atropine”; also “ATR”), sparteine, naringin, caffeic acid(“caffeine”;also “CAF”), quinacrine, and epicatechin. See Ming et al., 1999, Proc.Natl. Acad. Sci. U.S.A. 96:9903-9908, incorporated by reference herein.

In utilizing such cell systems, the cells expressing the TRP8 channelprotein are exposed to a test compound or to vehicle controls (e.g.,placebos). After exposure, the cells can be assayed to measure theexpression and/or activity of components of the signal transductionpathway of TRP8, or the activity of the signal transduction pathwayitself can be assayed.

The ability of a test molecule to modulate the activity of TRP8 may bemeasured using standard biochemical and physiological techniques.Responses such as activation or suppression of catalytic activity,phosphorylation or dephosphorylation of TRP8 and/or other proteins,activation or modulation of second messenger production, changes incellular ion levels, association, dissociation or translocation ofsignaling molecules, or transcription or translation of specific genesmay be monitored. In non-limiting embodiments of the invention, changesin intracellular Ca²⁺ levels may be monitored by the fluorescence ofindicator dyes such as indo, fura, etc. In addition activation of cyclicnucleotide phosphodiesterase, adenylate cyclase, phospholipases ATPasesand Ca²⁺ sensitive release of neurotransmitters may be measured toidentify compounds that modulate TRP8 signal transduction. Further,changes in membrane potential resulting from modulation of the TRP8channel protein can be measured using a voltage clamp or patch recordingmethods.

For example, after exposure to a test compound, cell lysates can beassayed for increased intracellular levels of Ca²⁺ and activation ofcalcium dependent down stream messengers such as phosphodiesterase,phospholipases, ATPases or cAMP. The ability of a test compound toincrease intracellular levels of Ca²⁺ and activate phosphodiesterase ordecrease cAMP levels compared to those levels seen with cells treatedwith a vehicle control, indicates that the test compound acts as anagonist (i.e., is a TRP8 activator) and induces signal transductionmediated by the TRP8 expressed by the host cell. The ability of a testcompound to inhibit bitter tastant induced calcium influx and inhibitphosphodiesterase or increase cAMP levels compared to those levels seenwith a vehicle control indicates that the test compound acts as anantagonist (i.e., is a TRP8 inhibitor) and inhibits signal transductionmediated by TRP8.

In a specific embodiment of the invention, levels of cAMP can bemeasured using constructs containing the cAMP responsive element linkedto any of a variety of different reporter genes. Such reporter genes mayinclude but are not limited to chloramphenicol acetyltransferase (CAT),luciferase, β-glucuronidase (GUS), growth hormone, or placental alkalinephosphatase (SEAP). Such constructs are introduced into cells expressingTRP8 channel protein thereby providing a recombinant cell useful forscreening assays designed to identify modulators of TRP8 activity.

Following exposure of the cells to the test compound, the level ofreporter gene expression may be quantitated to determine the testcompound's ability to regulate TRP8 activity. Alkaline phosphataseassays are particularly useful in the practice of the invention as theenzyme is secreted from the cell. Therefore, tissue culture supernatantmay be assayed for secreted alkaline phosphatase. In addition, alkalinephosphatase activity may be measured by calorimetric, bioluminescent orchemilumenscent assays such as those described in Bronstein, I. et al.(1994, Biotechniques 17: 172-177). Such assays provide a simple,sensitive easily automatable detection system for pharmaceuticalscreening.

Additionally, to determine intracellular cAMP concentrations, ascintillation proximity assay (SPA) may be utilized (SPA kit is providedby Amersham Life Sciences, Illinois). The assay utilizes ¹²⁵I-labelcAMP, an anti-cAMP antibody, and a scintillant-incorporated microspherecoated with a secondary antibody. When brought into close proximity tothe microsphere through the labeled cAMP-antibody complex, ¹²⁵I willexcite the scintillant to emit light. Unlabeled cAMP extracted fromcells competes with the ¹²⁵I-labeled cAMP for binding to the antibodyand thereby diminishes scintillation. The assay may be performed in96-well plates to enable high-throughput screening and 96 well-basedscintillation counting instruments such as those manufactured by Wallacor Packard may be used for readout.

In yet another embodiment of the invention, levels of intracellular Ca²⁺can be monitored using Ca²⁺ indication dyes, such as Fluo-3 and Fura-Redusing methods such as those described in Komuro and Rakic, 1998, In: TheNeuron in Tissue Culture. L.W. Haymes, Ed. Wiley, New York.

Test activators which activate the activity of TRP8, identified by anyof the above methods, may be subjected to further testing to confirmtheir ability to induce a bitterness perception. Test inhibitors whichinhibit the activation of TRP8 by bitter tastants, identified by any ofthe above methods, may then be subjected to further testing to confirmtheir inhibitory activity. The ability of the test compound to modulatethe activity of the TRP8 receptor may be evaluated by behavioral,physiologic, or in vitro methods.

For example, a behavioral study may be performed where a test animal maybe offered the choice of consuming a composition comprising the putativeTRP8 inhibitor and the same composition without the added compound. Apreference for the composition comprising a test compound, indicated,for example, by greater consumption, would have a positive correlationwith TRP8 inhibitory activity. Additionally, avoidance by a test animalof food containing a putative activator of TRP8 would have a positivecorrelation with the identification of an bitterness activator.

In addition to cell based assays, non-cell based assay systems may beused to identify compounds that interact with, e.g., bind to TRP8. Suchcompounds may act as antagonists or agonists of TRP8 activity and may beused to regulate bitter taste perception.

To this end, soluble TRP8 may be recombinantly expressed and utilized innon-cell based assays to identify compounds that bind to TRP8. Therecombinantly expressed TRP8 polypeptides or fusion proteins containingone or more of the domains of TRP8 prepared as described in Section 5.2,infra, can be used in the non-cell based screening assays. For example,peptides corresponding to one or more of the cytoplasmic ortransmembrane domains of TRP8, or fusion proteins containing one or moreof the cytoplasmic or transmembrane domains of TRP8 can be used innon-cell based assay systems to identify compounds that bind to thecytoplasmic portion of the TRP8; such compounds may be useful tomodulate the signal transduction pathway of the TRP8. In non-cell basedassays the recombinantly expressed TRP8 may be attached to a solidsubstrate such as a test tube, microtitre well or a column, by meanswell known to those in the art (see Ausubel et al., ura). The testcompounds are then assayed for their ability to bind to the TRP8.

The TRP8 channel protein may be one which has been fully or partiallyisolated from other molecules, or which may be present as part of acrude or semi-purified extract. As a non-limiting example, the TRP8channel protein may be present in a preparation of taste receptor cellmembranes. In particular embodiments of the invention, such tastereceptor cell membranes may be prepared as set forth in Ming, D. et al.,1998, Proc. Natl. Sci. U.S.A. 95:8933-8938, incorporated by referenceherein. Specifically, bovine circumvallate papillae (“taste tissue”,containing taste receptor cells), may be hand dissected, frozen inliquid nitrogen, and stored at −80° C. prior to use. The collectedtissues may then be homogenized with a Polytron homogenizer (threecycles of 20 seconds each at 25,000 RPM) in a buffer containing 10 mMTris at pH 7.5, 10% vol/vol glycerol, 1 mM EDTA, 1 mM DTT, 10 μg/μlpepstatin A, 10 μg/μl leupeptin, 10 μg/μl aprotinin, and 100 μM4-(2-amino ethyl) benzenesulfoyl fluoride hydrochloride. Afterparticulate removal by centrifugation at 1,500×g for 10 minutes, tastemembranes may be collected by centrifugation at 45,000×g for 60 minutes.The pelleted membranes may then be rinsed twice, re-suspended inhomogenization buffer lacking protease inhibitors, and furtherhomogenized by 20 passages through a 25 gauge needle. Aliquots may thenbe either flash frozen or stored on ice until use. As anothernon-limiting example, the taste receptor may be derived from recombinantclones (see Hoon, M. R. et al., 1999 Cell 96, 541-551).

Assays may also be designed to screen for compounds that regulate TRP8expression at either the transcriptional or translational level. In oneembodiment, DNA encoding a reporter molecule can be linked to aregulatory element of the TRP8 gene and used in appropriate intactcells, cell extracts or lysates to identify compounds that modulate TRP8gene expression. Appropriate cells or cell extracts are prepared fromany cell type that normally expresses the TRP8 gene, thereby ensuringthat the cell extracts contain the transcription factors required for invitro or in vivo transcription. The screen can be used to identifycompounds that modulate the expression of the reporter construct. Insuch screens, the level of reporter gene expression is determined in thepresence of the test compound and compared to the level of expression inthe absence of the test compound.

To identify compounds that regulate TRP8 translation, cells or in vitrocell lysates containing TRP8 transcripts may be tested for modulation ofTRP8 mRNA translation. To assay for inhibitors of TRP8 translation, testcompounds are assayed for their ability to modulate the translation ofTRP8 mRNA in in vitro translation extracts.

In addition, compounds that regulate TRP8 activity may be identifiedusing animal models. Behavioral, physiological, or biochemical methodsmay be used to determine whether TRP8 activation has occurred.Behavioral and physiological methods may be practiced in vivo. As anexample of a behavioral measurement, the tendency of a test animal tovoluntarily ingest a composition comprising the bitter tastant, in thepresence or absence of test inhibitor, may be measured. If the bittertastant activates TRP8 in the animal, the animal may be expected toexperience a bitter taste, which would discourage it from ingesting moreof the composition. If the animal is given a choice of whether toconsume a composition containing bitter tastant only (with activatedTRP8) or a composition containing bitter tastant together with abitterness inhibitor (with lower levels of activated TRP8), it would beexpected to prefer to consume the composition containing the bitternessinhibitor. Thus, the relative preference demonstrated by the animalinversely correlates with the activation of the TRP8 channel.

Physiological methods include nerve response studies, which may beperformed using a nerve operably joined to a taste receptor cellcontaining tissue, in vivo or in vitro. Since exposure to bitter tastantwhich results in TRP8 activation may result in an action potential intaste receptor cells that is then propagated through a peripheral nerve,measuring a nerve response to a bitter tastant is, inter alia, anindirect measurement of TRP8 activation. An example of nerve responsestudies performed using the glossopharyngeal nerve are described inNinomiya, Y., et al., 1997, Am. J. Physiol. (London) 272:R1002-R1006.

The assays described above can identify compounds which modulate TRP8activity. For example, compounds that affect TRP8 activity include butare not limited to compounds that bind to the TRP8, and either activatesignal transduction (agonists) or block activation (antagonists).Compounds that affect TRP8 gene activity (by affecting TRP8 geneexpression, including molecules, e.g., proteins or small organicmolecules, that affect transcription or interfere with splicing eventsso that expression of the full length or the truncated form of the TRP8can be modulated) can also be identified using the screens of theinvention. However, it should be noted that the assays described canalso identify compounds that modulate TRP8 signal transduction e.g.,compounds which affect downstream signaling events, such as inhibitorsor enhancers of G protein activities which participate in transducingthe signal activated by tastants binding to their receptor). Theidentification and use of such compounds which affect signaling eventsdownstream of TRP8 and thus modulate effects of TRP8 on the perceptionof taste are within the scope of the invention.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to, small organic or inorganic compounds,peptides, antibodies and fragments thereof, and other organic compounds(e.g, peptidomimetics) that bind to TRP8 and either mimic the activitytriggered by the natural tastant ligand (i.e., agonists) or inhibit theactivity triggered by the natural ligand (i.e., antagonists).

Compounds may include, but are not limited to, peptides such as, forexample, soluble peptides, including but not limited to members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghten, R. et al., 1991, Nature 354:84-86); andcombinatorial chemistry-derived molecular library made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; (see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope bindingfragments thereof), and small organic or inorganic molecules.

Other compounds which may be screened in accordance with the inventioninclude but are not limited to small organic molecules that affect theexpression of the TRP8 gene or some other gene involved in the TRP8signal transduction pathway e.g., by interacting with the regulatoryregion or transcription factors involved in gene expression); or suchcompounds that affect the activity of the TRP8 or the activity of someother intracellular factor involved in the TRP8 signal transductionpathway, such as, for example, a TRP8 associated G-protein.

5.6. COMPOSITIONS CONTAINING MODULATORS OF TRP8 AND THEIR USES

The present invention provides for methods of inhibiting a bitter tasteresulting from contacting a taste tissue of a subject with a bittertastant, comprising administering to the subject an effective amount ofa TRP8 inhibitor, such as a TRP8 inhibitor identified by measuring TRP8activation as set forth in Section 5.5 supra. The present invention alsoprovides for methods of inhibiting a bitter taste of a composition,comprising incorporating, in the composition, an effective amount of aTRP8 inhibitor. An “effective amount” of the TRP8 inhibitor is an amountthat subjectively decreases the perception of bitter taste and/or thatis associated with a detectable decrease in TRP8 activation as measuredby one of the above assays.

The present invention further provides for a method of producing theperception of a bitter taste by a subject, comprising administering, tothe subject, a composition comprising a compound that activates TRP8activity such as a bitterness activator identified as set forth inSection 5.5 supra. The composition may comprise an amount of activatorthat is effective in producing a taste recognized as bitter by asubject.

Accordingly, the present invention provides for compositions comprisingbitterness activators and bitterness inhibitors. Such compositionsinclude any substances which may come in contact with taste tissue of asubject, including but not limited to foods, beverages, pharmaceuticals,dental products, cosmetics, and wetable glues used for envelopes andstamps.

In one set of embodiments, a bitterness inhibitor is used to counteractthe perception of bitterness associated with a co-present bittertastant. In these embodiments, a composition of the invention comprisesa bitter tastant and a bitterness inhibitor, where the bitternessinhibitor is present at a concentration which inhibits bitter tasteperception. For example, when the concentration of bitter tastant in thecomposition and the concentration of bitterness inhibitor in thecomposition are subjected to an assay as disclosed in Section 5.1 supra,the bitterness inhibitor inhibits the activation of TRP8 by the bittertastant.

The present invention may be used to improve the taste of foods bydecreasing or eliminating the aversive effects of bitter tastants. If abitter tastant is a food preservative, the TRP8 inhibitors of theinvention may permit or facilitate its incorporation into foods, therebyimproving food safety. For foods administered as nutritionalsupplements, the incorporation of TRP8 inhibitors of the invention mayencourage ingestion, thereby enhancing the effectiveness of thesecompositions in providing nutrition or calories to a subject.

The TRP8 inhibitors of the invention may be incorporated into medicaland/or dental compositions. Certain compositions used in diagnosticprocedures have an unpleasant taste, such as contrast materials andlocal oral anesthetics. The TRP8 inhibitors of the invention may be usedto improve the comfort of subjects undergoing such procedures byimproving the taste of compositions. In addition, the TRP8 inhibitors ofthe invention may be incorporated into pharmaceutical compositions,including tablets and liquids, to improve their flavor and improvepatient compliance (particularly where the patient is a child or anon-human animal).

The TRP8 inhibitors of the invention may be comprised in cosmetics toimprove their taste features. For example, but not by way of limitation,the TRP8 inhibitors of the invention may be incorporated into facecreams and lipsticks. In addition, the TRP8 inhibitors of the inventionmay be incorporated into compositions that are not traditional foods,beverages, pharmaceuticals, or cosmetics, but which may contact tastemembranes. Examples include, but are not limited to, soaps, shampoos,toothpaste, denture adhesive, glue on the surfaces of stamps andenvelopes, and toxic compositions used in pest control (e.g., rat orcockroach poison).

6. EXAMPLE CLONING AND CHARACTERIZATION OF THE TRP8 GENE

This following subsection describes the isolation and characterizationof a transient receptor protein channel referred to as TRP8. The deducedamino acid sequence of TRP8 shows homology with other TRP proteins.Northern Blot analysis indicates high level expression of TRP8 RNA intaste receptor cells.

6.1. MATERIALS AND METHODS 6.1.1 CLONING OF THE TRP8 GENE

Single cell reverse transcription-polymerase chain reaction (RT-PCR) anddifferential screening were used to clone genes specifically orselectively expressed in the subset of taste receptor cells that arepositive for expression of the G protein gustducin. Individualgustducin-positive cells were isolated from mouse circumvallate papillae(Huang et al. 1999 Nature Neuroscience 2: 1055-1062). The mRNAs fromindividual cells were reverse transcribed into cDNA followed by PCRamplification. Multiple cDNA libraries from single taste receptor cellswere constructed by subcloning the amplified cDNAs into bacteriophagevectors. The cDNA libraries were analyzed by differential screening withself-probe (P³²-labelled amplified cDNAs from the same cell) andnon-self probe (P³²-labeled amplified cDNAs from another taste cell).Hybridization was carried out at 65° C. for 20 hours in 0.5 M sodiumphosphate buffer (pH 7.3) containing 1% bovine serum albumin and 4% SDS.The membranes were washed twice at 65° C. in 0.1% SDS, 0.5×SSC for 20minutes and one time at 65° C. in 0.1% SDS, 0.1×SSC for 15 minutes. Themembranes were exposed to X-ray film at −80° C. with an intensifyingscreen for three days. Clones which more strongly hybridize to selfprobe than to non-self probe were isolated and their inserts sequenced.

Using this clone as a probe (LQSEQ91), a mouse taste tissue cDNA librarywas screened for full-length clones under the same hybridizationconditions as specified above. Sequencing the clones containing thelongest inserts produced a full-length clone with greatest similarity toa family of calcium channel proteins known as transient receptorpotential (TRP) channels.

25 μg of total RNA was isolated by acid guanidiniumthiocyanate/phenol/chloroform extraction (P. Chromczynski and N. Sacchi,1987, Anal. Biochem. 162:156) from the following mouse tissues: tastebud enriched epithelium, nontaste lingual epithelium, brain, retina,olfactory epithelium, stomach, small intestine, liver, spleen, kidney,lung, heart, thymus, uterus, testis and skeletal muscle. The RNAs wereelectrophoresed on 1.5% agarose gel containing 6.7% formaldehyde,transferred and fixed to a nylon membrane by UV irradiation. The blotwas hybridized with a radiolabeled 1.7 kb fragment generated from the3′-end of mouse TRP 8 cDNA by random priming with Exo(−) Klenowpolymerase in the presence of (∝-³²P)-dCTP. The hybridization wascarried out in 0.25 M sodium phosphate buffer (pH 7.2) containing 7% SDSat 65° C. with agitation for 24 hours. The membrane was washed twice in20 mM sodium phosphate buffer (pH 7.2) containing 5% SDS at 65° C. for40 minutes and twice in the same buffer containing 1% SDS at 65° C. for40 minutes. The blot was exposed to X-ray film at −80° C. with anintensifying screen for 5 days.

A BLAST search of human high throughput DNA sequences and genomicsequences was done using the mouse TRP8 sequence as the query. From thissearch a BAC clone was identified that contained the entire human TRP8gene. The Genscan program was then used to identify the predictedprotein-coding exons of the human TRP8 gene. The regions were alignedwith the mouse TRP8 cDNA to refine the predicted human TRP8 codingregion, leading to deduction of the entire human coding region.

6.1.2. NORTHERN HYBRIDIZATION

Total RNAs were isolated from several mouse tissues using the Trizolreagents, then 25 μg of each RNA was electrophoresed per lane on a 1.5%agarose gel containing 6.7% formaldehyde. The samples were transferredand fixed to a nylon membrane by UV irradiation. The blot wasprehybridized at 65° C. in 0.25 M sodium phosphate buffer (pH 7.2)containing 7% SDS and 40 μg/ml herring sperm DNA with agitation for 5hours; hybridization for 20 hours with the ³²P-radiolabeled mouse TRP8probe was carried out in the same solution. The membrane was washedtwice at 65° C. in 20 mM sodium phosphate buffer (pH 7.2) containing 5%SDS for 40 minutes, twice at 65° C. in the same buffer containing 1% SDSfor 40 minutes, and once at 70° C. in 0.1×SSC and 0.1% SDS for 30minutes. The blot was exposed to X-ray film for 3 days at −80° C. withdual intensifying screens. The ³²P-labeled TRP8 probe was generated byrandom nonamer priming of a0.48 kb cDNA fragment of TRP8 correspondingto the 3′-UTR sequence using Exo(−) Klenow polymerase in the presence of(α-³²P)-dCTP.

6.1.3. IN SITU HYBRIDIZATION

³³P-labeled RNA probes [TRP8 (1.7 kb) and α-gustducin (1 kb)] were usedfor in situ hybridization of frozen sections (10 μm) of mouse lingualtissue. Hybridization and washing were as described (Asano-Miyoshi etal., 2000, Neurosci Lett 283:61-4). Slides were coated with Kodak NTB-2nuclear track emulsion and exposed at 4° C. for 3 weeks and thendeveloped and fixed.

6.1.4. IMMUNOCYTOCHEMISTRY

Polyclonal antisera against a keyhole limpet hemocyanin-conjugated TRP8peptide (aa 1028-1049) were raised in rabbits. The PLCβ2 antibody wasobtained from Santa-Cruz Biotechnologies; the anti-β-gustducin andanti-Gγ13 antibodies were as described (Ruiz-Avila et al., 1995, Nature376:80-5; Huang et al., 1999, Nat Neurosci 2:1055-62). Ten micron thickfrozen sections of murine lingual tissue (previously fixed in 4%paraformaldehyde and cryoprotected in 20% sucrose) were blocked in 3%BSA, 0.3% Triton X-100, 2% goat serum and 0.1% Na Azide in PBS for 1hour at room temperature and then incubated for 8 hours at 4° C. withpurified antibody against α-gustducin, or antiserum against TRP8(1:800). The secondary antibodies were Cy3-conjugated goat-anti-rabbitIg for TRP8 and fluorescein-conjugated goat-anti-rabbit Ig for PLCβ2,α-gustducin or Gγ13. TRP8 immunoreactivy was blocked by preincubation ofthe antisera with the immunizing peptides at 20 μM. Preimmune serum didnot show any immunoreactivity. Sections were double-immunostained withTRP8 plus one of the following antibodies: anti-PLCβ2, anti-α-gustducinor anti-Gγ13. The sections were incubated sequentially with TRP8antiserum, anti-rabbit-Ig-Cy3 conjugate, normal anti-rabbit-Ig,anti-PLCβ2 (or anti-α-gustducin or anti-Gγ13) antibody and finally withanti-rabbit-Ig-FITC conjugate with intermittent washes between eachstep. Control sections that were incubated with all of the above exceptanti-PLCβ2 (or anti-α-gustducin or anti-Gγ13) antibody did not show anyfluorescence in the green channel.

6.1.5. GENE EXPRESSION PROFILING

Single taste receptor cell RT-PCR products (5 μl) were fractionated bysize on a 1.6% agarose gel and transferred onto a nylon membrane. Theexpression patterns of the isolated cells were determined by Southernhybridization with 3′-end cDNA probes for mouse TRP8, α-gustducin, Gβ3,Gγ13, PLCβ2 and G3PDH. Blots were exposed for five hours at −80° C.Total RNAs from a single circumvallate papilla and a similar-sized pieceof non-gustatory epithelium were also isolated, reverse transcribed,amplified and analyzed as for the individual cells.

6.1.6. HETEROLOGOUS EXPRESSION

Oocytes were injected with 50 ng of TRP8 cRNA. 48 hours after injectionoocytes were incubated in Thapsigargin (2 μM) and X-Rhod-1-AM (the Ca⁺⁺sensing dye) for 3 hours at room temperature.

6.2. RESULTS 6.2.1. IDENTIFICATION OF A NOVEL TRP CHANNEL IN TASTE CELLS

Using single cell reverse transcription-polymerase chain reaction, aclone was isolated that was expressed in gustducin-positive cells butabsent from gustducin-negative cells. A search of the expressed sequencetag (EST) dbest database found no matches, suggesting that this clone'spattern of expression is highly restricted to tissues not generallyfound in EST databases, such as taste tissue.

Using this clone as a probe, a mouse taste tissue cDNA library wasscreened for full-length clones. Sequencing the clones containing thelongest inserts produced a full-length clone with the sequence indicatedin FIG. 1. The deduced amino acid sequence of the cDNA clone is shown inFIG. 2.

The isolated cDNA showed the greatest similarity to a family of calciumchannel proteins known as transient receptor potential (TRP) channels.The similarity of the isolated clone to this family of proteinsindicated that a TRP channel had been identified. Currently seven TRPchannels are known to exist, making this clone the eighth member, namedby convention TRP8. Mouse TRP8 (TRP8) is most closely related to TRP7with an identity at the amino acid level of 40%. The predictedtopography of the TRP8 channel inserted within the cell membrane ispresented in FIGS. 6A-C.

Based upon homology of the mouse clone with a region of human chromosome11p15.5 contained in a BAC clone (genebank #AC003693) a human TRP8ortholog was identified. The nucleotide sequence of the human TRP8 gene,as well as the deduced amino acid sequence, are depicted in FIGS. 3A-B(SEQ ID NO: 3) and 4 (SEQ ID NO: 4), respectively. A comparison of themurine and human TRP 8 proteins is shown in FIG. 5 (SEQ ID NO: 2 and SEQID NO: 4, respectively). This region of human chromosome 11p15.5 issyntenic with the distal region of mouse chromosome 7. In both cases,TRP8 and hTRP8 map between genes for Kvlqt1 and TSSC4.

6.2.2. TRP8 IS SELECTIVELY EXPRESSED IN TASTE TISSUE

Although TRP8 was identified during a differential screen ofα-gustducin-positive (+) vs. α-gustducin (−)⁻ TRCs, it was possible thatTRP8 might be more broadly expressed in other taste cells and/ortissues. To determine the tissue distribution of TRP8 mRNA a northernblot with multiple murine tissues was carried out. An TRP8 3′-UTR probehybridized predominantly to a transcript of 4.5 kb in taste tissue, withno detectable expression in control non-taste tissue. Moderateexpression was detected in stomach and small intestine; weak expressionwas noted in uterus and testis (FIG. 7A). This is in contrast to theresults of Enklaar et al., (2000, Genomics 67:179-87). Using anRT-PCR-generated probe designed to amplify the 3′ portion of TRP8'scoding region they detected highest expression in liver and low levelexpression in other peripheral tissues (e.g. heart, brain, kidney andtestis). Their RT-PCR probe may have detected by cross-hybridizationother TRP8 mRNAs or an alternatively spliced MRNA with a different3′-end from that present in our 3′-UTR probe. As an independent measureof expression of TRP8, we carried out western blots using an anti-TRP8antibody (FIG. 7B). TRP8 protein of the predicted molecular weight (˜130kDa) was detected in stomach and small intestine; a species of higherthan expected molecular weight was identified in liver and kidney andmay represent either an TRP8 -related protein or an TRP8 product from analternatively spliced message.

6.2.3. TRP8 IS EXPRESSED IN PARTICULAR SUBSETS OF TASTE RECEPTOR CELLS

In situ hybridization was used to determine the cellular pattern ofexpression of TRP8 in mouse TRCs. TRP8 mRNA was observed in TRCs incircumvallate and foliate papillae, but not in the surroundingnon-gustatory epithelia (FIG. 8). TRP8⁺ TRCs were present in themajority of the taste buds, although not all TRCs were positive,suggesting restricted expression to a subset of TRCs. The generalpattern of TRP8 expression was comparable to that of α-gustducin,although the α-gustducin signal was somewhat more intense (FIG. 8D).Controls with sense probes showed minimal non-specific hybridization totaste tissue with either the TRP8 probe (FIG. 8B) or the (α-gustducinprobe (FIG. 8E).

To determine if TRP8 is co-expressed in TRCs with signal transductionelements that might be involved in its activation, we performed singleand double immunohistochemistry of TRC-containing tissue sections. TRP8protein was co-expressed absolutely with Gγ13 (FIG. 9ABC) and PLCβ2(FIG. 9GHI), suggesting that these three molecules might be part of acommon signal transduction pathway. TRP8 co-expressed largely, but notabsolutely, with (α-gustducin (FIG. 9DEF): a subset of the TRP8⁺ TRCswere negative for α-gustducin, although all α-gus⁺ TRCs were positivefor TRP8. This pattern is consistent with our observations that α-gus⁺TRCs constitute a subset of TRCs that are positive for Gγ13, Gβ1, PLCβ2and IP₃R3 (Huang et al, 1999,). The slight differences in distributionat the cellular level among the different molecules could be explainedby the different topologies that each protein displays: TRP8 is anintegral membrane protein, whereas α-gustducin and PLCβ2 aremembrane-associated proteins. The expression of human TRP8 (hTRP8 ) inhuman fungiform taste buds was also confirmed.

To independently monitor co-expression of TRP8 in TRCs with theabove-mentioned signal transduction elements, as well as with Gβ1 andGβ3, a single cell expression profiling was carried out (Huang et al.,1999, Nat Neurosci 2:1055-62)). In this way it was determined thatexpression of α-gustducin, Gβ1, Gβ3, Gγ13, PLCβ2 and TRP8 was restrictedto taste tissue (FIG. 10, left panel), and that in this set of 24 TRCs,TRP8 co-expressed absolutely with α-gustducin, Gβ3, Gγ13, PLCβ2 (FIG.10, right panel); expression of TRP8 also overlapped in large part withthat of Gβ1 (15 of 19 TRP8⁺ cells were also Gβ1⁺). The coincidentexpression of these various signal transduction molecules with TRP8could provide the physical opportunity for activation of TRP8 by IP₃ (byactivation of IP₃ receptors) or DAG (by direct activation of TRP8)generated by a signaling pathway in which GPCRs coupled toheterotrimeric gustducin (i.e. α-gustducin/β3/γ13) or to otherGα/β1,β3/γ13-containing heterotrimers might release βγ to activatePLCβ2. Consistent with this is the recent identification in TRCs of IP₃receptor subtype III (IP₃R3), and the demonstration that IP₃R3co-localizes in large part with α-gustducin, Gγ13 and PLCβ2.

6.2.4. OTHER TRP FAMILY MEMBERS ARE NOT DETECTABLY EXPRESSED IN TASTETISSUE

Native TRP channels are thought to form homo- and hetero-multimers. Toidentify potential partners for TRP8 in TRCs PCR was used to determineif murine TRP channels 1-6 (TRP 1-6) are expressed in taste tissue(brain tissue provided a positive control). Amplification by the PCRusing primer pairs specific for TRP 1-6 identified products of thecorrect size for all six TRP family members when brain cDNA was used asthe template (FIG. 11, lower panel); DNA sequencing of these productsconfirmed amplification of all six TRP family members. TRP8 was notamplified when brain cDNA was the template (FIG. 11, lower panel),although it was amplified when taste cDNA provided the template (FIG.11, upper panel) (amplification of TRP8 was confirmed by DNAsequencing). None of the other six TRP family members were amplifiedwhen taste tissue cDNA was used as the template (FIG. 11, upper panel),suggesting that they are not highly expressed, if at all, in TRCs. In aseparate experiment using TRP7 specific primers, TRP7 was detected byPCr in brain cDNA, but not in taste cDNA. Novel TRP channels beyondthese seven members might be expressed in TRCs, but at the present timeit would appear that TRP8 is the only known TRP channel highly expressedin taste tissue, and as shown above, in TRCs.

6.2.5. EXPRESSED TRP8 ACTS AS A STORE OPERATED CHANNEL

To determine if TRP8 can function as a calcium channel, TRP8 wasexpressed in Xenopus oocytes. The oocytes possess an endogenouscalcium-activated chloride conductance (ICl_(Ca)) that may be used tomonitor Ca⁺⁺ influx due to activation of store operated Ca⁺⁺ channelsbelonging to the TRP family. TRP8 RNA obtained by in vitro transcriptionwas injected into Xenopus oocytes and two electrode voltage clamprecordings were performed two days later. To induce depletion ofinternal Ca⁺⁺ stores, oocytes were incubated for 2 hours before therecording in 2 μM thapsigargin (TPN), an irreversible inhibitor of thesarco(endo)plasmic reticulum Ca⁺⁺-ATPase (SERCA).

Representative recording traces of oocytes injected with TRP8 RNA andtreated with TPN demonstrated a robust and distinct inward currentelicited by the addition of Ca⁺⁺ to the external bath (FIG. 12A). Thesetraces differ dramatically from those of control oocytes injected withwater (FIG. 12B), indicating that TRP8 encodes a functional Ca⁺⁺ channelwhose activation is dependent on the filling status of the internal Ca⁺⁺stores (compare FIG. 12 panels A and B), and whose function relies onthe availability of external Ca⁺⁺. The control oocytes express anendogenous TRP channel (XTrp) (Bobanovic et al., 1999, Biochem J.340:593-9)) that can be activated by TPN treatment (FIG. 12B). Analysisof the total inward current (FIG. 12D) generated under conditions whenCa⁺⁺ is present in the extracellular medium clearly demonstrated theeffect of TRP8 expression. To confirm that TRP8 protein was actuallyexpressed in the oocytes, we carried out a western blot of the membraneproteins from TRP8 RNA-injected oocytes using an anti-TRP8 antibody: a130 kDa protein of the expected size was detected.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Various references are cited herein, the disclosures ofwhich are incorporated by reference in their entireties.

1. A method for identifying a compound that potentially induces theperception of a bitter taste comprising: (i) contacting an isolated cellexpressing the TRP8 channel protein of SEQ ID NO: 4 with a test compoundand measuring the level of TRP8 activation; (ii) in a separateexperiment, contacting an isolated cell expressing the TRP8 channelprotein of SEQ ID NO: 4 with a vehicle control and measuring the levelof TRP8 activation where the conditions are essentially the same as inpart (i); and (iii) comparing the level of activation of TRP8 measuredin part (i) with the level of activation of TRP8 in part (ii), whereinthe level of TRP8 activation is measured by measuring the membranepotential of the cell and wherein an increased level of activated TRP8in the presence of the test compound indicates that the test compoundpotentially induces the perception of a bitter taste.
 2. The methodaccording to claim 1, wherein said measuring the level of TRP8activation is carried out with one or more fluorescence-indicator dyes.3. The method according to claim 1, wherein said measuring the membranepotential of the cell is carried out under voltage clamp assayconditions.
 4. The method according to claim 1, wherein said measuringthe membrane potential of the cell is carried out under patch recordingassay conditions.